Following Molecular Transitions with Single Residue Spatial and Millisecond Time Resolution

"Footprinting" describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions, respectively. The hydroxyl radical (•OH) is a uniquely insightful footprinting probe by virtue of it being among the most reactive chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved •OH footprinting is presented based on the Fenton reaction, Fe(II) + H₂O₂ → Fe(III) + •OH + OH^-. It is implemented using a standard three-syringe quench-flow mixer. The utility of this method is demonstrated by its application to the studies on RNA folding. Its applicability to a broad range of biological questions involving the function of DNA, RNA, and proteins is discussed.